September 1979
FEBS LETTERS
Volume 105, number I
COMPACT
GLOBULAR
STRUCTURE
FROM ESCHERICHIA
OF PROTEIN
S15
COLI RIBOSOMES
Z. V. GOGIA, S. Yu. VENYAMINOV, V. N. BUSHUEV*, I. N. SERDWK, V. I. LIM and A. S. SPIRIN of Protein Research, USSR Academy of Sciences and *Institute of Biological Physics, USSR Academy of Sciences,
Institute
Poustchino, Moscow Region, USSR
Received 7 June 1979
1. Introduction The ribosomal protein S15 is one of the 2 1 proteins of the small (30 S) subunit of the Escherichia coZi ribosomes [ 11. Its polypeptide chain consists of only 87 amino acid residues [2]. It became of special interest to study the conformation of protein S15 when it was claimed [3] that its antigenic determinants in the 30 S subunit were revealed by immunoelectron microscopy at the most distal sites of the particle (-200 A apart), thus suggesting its extremely elongated or even fibrous conformation. Recently the concept of elongated, non-globular conformations of ribosomal proteins has become widely accepted (e.g., review [4] ). A number of reports have appeared where measurements of physical parameters of isolated ribosomal proteins in solution were used as evidence for their expanded conformations. In particular, both hydrodynamic [5] and small-angle X-ray scattering [6] measurements of protein S15 in solution indicated that the axial ratio of its molecules is about 5 : 1 or 6: 1 and that the molecular length is -100 A. Recently in our group direct data were obtained showing that the ribosomal proteins S4, S7, S8 and S16, when prepared carefully, have compact globular conformations in solution [7], in contrast to a number of reports about their elongated shapes. This has forced us to undertake a special complex study of protein S15 which is usually assumed to be a typical elongated protein of the ribosome. Circular dichroism, proton magnetic resonance and neutron scattering measurements of protein S15 in solution have shown that it is a compact globular molecule with a high content of secondary structure, a well developed ElsevierfNorth-Holland
Biomedical Press
tertiary structure and an almost spherical shape. On the basis of a theoretical stereochemical analysis of its primary structure, a model of the tertiary structure of protein S15 is proposed and discussed.
2. Materials and methods 2.1. Preparation of the ribosomal protein S1.5 The 30 S subunits of Escherichia coli MRE-600 ribosomes were isolated by sucrose gradient centrifugation in the presence of 0.5 M NHaCI and 1 mM MgClz [8] using a zonal rotor [9]. The 30 S particles were treated with 3 M LiCl in the presence of 5 mM MgClz in order to obtain the protein-deficient derivatives (core particles) retaining mainly the proteins S4, S7, S8, S15 and S16 [IO]. Protein S15 was extracted from the ‘core particles’ by 4 M urea with 3 M LiCl and then purified by phosphocellulose column chromatography in the presence of 6 M urea
[ill. The fraction of protein S15 eluted from the column was concentrated by Amicon ultracentrifugation using a UM-2 filter,and then re-chromatographed on Sephadex G-100 [ 111. The protein solution was kept frozen at -80°C. Immediately before experiments the solution was thawed and dialyzed in the cold, first against 2 changes of 1 M KC1 with 50 mM potassium phosphate (pH 5.6) then against several (5 or 6) changes of the standard solvent, 0.1 M NaCl-30 mM sodium phosphate (pH 5.6); if required, dialysis was continued against 3-6 changes of 0.1 M NaCl with 30 mM sodium phosphate prepared in 92% D20 (for neutron scattering), 99.8% DzO (for PMR 63
Volume
105, number
1
FEBS LETTERS
measurements) pD 5.3 (standard D20 solvents). The final solution of the renatured protein S15 was clarified by centrifugation at 16 000 rev./min for 30 min. The identity, purity and homogeneity of the protein Sl,5 preparations were checked by twodimensional gel electrophoresis in urea [ 121, onedimensional gel electrophoresis in SDS [ 131, N-terminal group determination [ 141, and amino acid analysis. The molecular weight of protein S15, determined by sedimentation equilibrium method [ 151 in the standard solvent, was 12 500 + 1000. 2.2. Absorption and CD spectra Measurements of absorption spectra were performed in an EPS3T Hitachi instrument. In order to estimate the extinction coefficient, the microtechnique of nitrogen determination [ 161 was used, assuming a nitrogen content of 19.7% [2] and introducing the correction for solution turbidity
[171. CD spectra were measured in a J41A JASCO instrument. Calibration of the instrument was done according to [ 181. The measurements were performed in cells 0.093,0.186,0.5 and 10.0 mm thick, in the far and near ultraviolet regions. For calculation of the ellipticity the mean molecular weight of an amino acid residue (MRW) was assumed to be 115.0 [2]. The estimation of the secondary structure content was done according to [ 191, using reported reference spectra [20].
September
1979
from the slope of the scattering curve in the Guinier coordinates (log I versus p2). The molecular weight was calculated from the curves as in [7].
3. Results 3 .l . Extinction coefficient and secondary structure Figure 1 presents the absorption spectrum of protein S15 in the ultraviolet region recorded in standard solvent. The extinction coefficient at 277 nm (A; “,p’“‘) is found to be 0.325 (*O.OlO). This extinction coefficient was used in all other experiments for determinations and checks of the protein concentration. It has been shown in special experiments that the absorption spectrum and the extinction coefficient do not change in solvents with pH values from 5.6 to 7 and salt concentration from 30 mM to 0.4 M. Figure 2 presents the CD spectrum of protein S15. It is seen that the spectrum corresponds to a highly a-helical protein. Estimation of the a-helical content in protein S15 from the CD spectrum [ 19,201 gives a value of 78%. The value of the helical content has been shown to remain unchanged, at least in the pH range 5.6 to 7 and salt concentration from 30 mM to 0.4 M. 3.2. Tertiary structure Figure 3 shows the PMR spectra of protein S15 in standard solvent (A) and in the presence of 5 M urea
2.3. PMR spectra Proton NMR spectra were recorded on a Bruker WH 360 spectrometer operating in the Fourier transform mode, using a pulse-length of 6 /LSwith 1.8 s intervals; the number of accumulations was 2000. Chemical shifts were measured relative to sodium 2,2-dimethyl-2-silapentane sulphonate as an internal standard. Spectra were obtained in 5 mm tubes with 0.8-I .O mg protein/ml in standard D20 solvent. 2.4. Neutron scattering Neutron scattering experiments were done in the Institut Laue-Langevin, Grenoble, on the high-flux reactor using the Dll camera [21] as in [7]. The cell was 2 mm thick with 0.75 mg protein/ml in standard DzO solvent. The radius of gyration was calculated 64
240
260
280
300
Wavelength,
320
340
360
nm
Fig.1. Absorption spectrum of protein S15 in the near ultraviolet region. Solvent: 0.1 M NaCl, 30 mM sodium phosphate (pH 5.6).
FEBSLETTERS
Volume 105, number 1
Wavelength,
September 1979
the spectrum of the protein under non-denaturing conditions (fig3A) contains wide overlapping resonance lines, mainly of apolar aliphatic residues and threonines. This indicates that the aliphatic side groups and the threonine residues are in different chemical surroundings within the protein, suggesting a specific folding of its chain. In the extreme high-field region, between 0.8 and 0.5 ppm, the PMR spectrum of protein S15 has a number of resonance lines with chemical shifts which are absent in individual amino acid residues [22]. These resonances can be attributed to aliphatic amino acid residues located near to aromatic amino acids in a globular structure [22,23]. In the low-field region, between 6.5 and 8.5 ppm, the resonance lines of 4 histidine, 2 tyrosine and 2 phenylalanine residues are revealed. Four separate signals, 2 protons each (at 6.79,6.85,7.09 and 7.14 ppm), have been attributed to the 2 tyrosine residues since they underwent a shift into the high field at pH >9.5 (data not shown). Inasmuch as the resonance lines at 6.79 and 7.09 ppm of one of the tyrosine residues are broader and their resolution poorer than the resonances at 6.85 and 7.14 ppm of the other, it can be suggested that one of the residues rotates around the Cfl-0 bond more freely than does the other [24].
nm
Wavelength, nm Fig.2. CD spectrum of protein S15 in the near (insertion) and the far ultraviolet regions. Solvent: 50 mM sodium phosphate (pH 5.6).
(B). Comparison of these spectra provides evidence that a well developed tertiary structure exists in protein S15 in standard solvent. In the high-field region, between 0.9 and 3.5 ppm,
His
C&i)
rn
7 mm
,
1
I
I
3
2
I
0
Fig.3. PMR spectra of protein S15. Solvent: 99.8% D,O with 0.1 M NaCl-30 mM sodium phosphate (pD 5.3) (A), and the same solvent in the presence of 5 M urea (B).
65
Volume 105, number 1
FEBS LETTERS
Protons at CZ of the imidazole rings of the 4 histidine residues of protein S15 under non-denaturing conditions exhibit separate signals in the region from 8.4 to 8.6 ppm indicating their different chemical surroundings, i.e., specific chain folding. Since their width is
FEBS LETTERS
Volume 105, number I
COMPACT
GLOBULAR
STRUCTURE
FROM ESCHERICHIA
OF PROTEIN
S15
COLI RIBOSOMES
Z. V. GOGIA, S. Yu. VENYAMINOV, V. N. BUSHUEV*, I. N. SERDWK, V. I. LIM and A. S. SPIRIN of Protein Research, USSR Academy of Sciences and *Institute of Biological Physics, USSR Academy of Sciences,
Institute
Poustchino, Moscow Region, USSR
Received 7 June 1979
1. Introduction The ribosomal protein S15 is one of the 2 1 proteins of the small (30 S) subunit of the Escherichia coZi ribosomes [ 11. Its polypeptide chain consists of only 87 amino acid residues [2]. It became of special interest to study the conformation of protein S15 when it was claimed [3] that its antigenic determinants in the 30 S subunit were revealed by immunoelectron microscopy at the most distal sites of the particle (-200 A apart), thus suggesting its extremely elongated or even fibrous conformation. Recently the concept of elongated, non-globular conformations of ribosomal proteins has become widely accepted (e.g., review [4] ). A number of reports have appeared where measurements of physical parameters of isolated ribosomal proteins in solution were used as evidence for their expanded conformations. In particular, both hydrodynamic [5] and small-angle X-ray scattering [6] measurements of protein S15 in solution indicated that the axial ratio of its molecules is about 5 : 1 or 6: 1 and that the molecular length is -100 A. Recently in our group direct data were obtained showing that the ribosomal proteins S4, S7, S8 and S16, when prepared carefully, have compact globular conformations in solution [7], in contrast to a number of reports about their elongated shapes. This has forced us to undertake a special complex study of protein S15 which is usually assumed to be a typical elongated protein of the ribosome. Circular dichroism, proton magnetic resonance and neutron scattering measurements of protein S15 in solution have shown that it is a compact globular molecule with a high content of secondary structure, a well developed ElsevierfNorth-Holland
Biomedical Press
tertiary structure and an almost spherical shape. On the basis of a theoretical stereochemical analysis of its primary structure, a model of the tertiary structure of protein S15 is proposed and discussed.
2. Materials and methods 2.1. Preparation of the ribosomal protein S1.5 The 30 S subunits of Escherichia coli MRE-600 ribosomes were isolated by sucrose gradient centrifugation in the presence of 0.5 M NHaCI and 1 mM MgClz [8] using a zonal rotor [9]. The 30 S particles were treated with 3 M LiCl in the presence of 5 mM MgClz in order to obtain the protein-deficient derivatives (core particles) retaining mainly the proteins S4, S7, S8, S15 and S16 [IO]. Protein S15 was extracted from the ‘core particles’ by 4 M urea with 3 M LiCl and then purified by phosphocellulose column chromatography in the presence of 6 M urea
[ill. The fraction of protein S15 eluted from the column was concentrated by Amicon ultracentrifugation using a UM-2 filter,and then re-chromatographed on Sephadex G-100 [ 111. The protein solution was kept frozen at -80°C. Immediately before experiments the solution was thawed and dialyzed in the cold, first against 2 changes of 1 M KC1 with 50 mM potassium phosphate (pH 5.6) then against several (5 or 6) changes of the standard solvent, 0.1 M NaCl-30 mM sodium phosphate (pH 5.6); if required, dialysis was continued against 3-6 changes of 0.1 M NaCl with 30 mM sodium phosphate prepared in 92% D20 (for neutron scattering), 99.8% DzO (for PMR 63
Volume
105, number
1
FEBS LETTERS
measurements) pD 5.3 (standard D20 solvents). The final solution of the renatured protein S15 was clarified by centrifugation at 16 000 rev./min for 30 min. The identity, purity and homogeneity of the protein Sl,5 preparations were checked by twodimensional gel electrophoresis in urea [ 121, onedimensional gel electrophoresis in SDS [ 131, N-terminal group determination [ 141, and amino acid analysis. The molecular weight of protein S15, determined by sedimentation equilibrium method [ 151 in the standard solvent, was 12 500 + 1000. 2.2. Absorption and CD spectra Measurements of absorption spectra were performed in an EPS3T Hitachi instrument. In order to estimate the extinction coefficient, the microtechnique of nitrogen determination [ 161 was used, assuming a nitrogen content of 19.7% [2] and introducing the correction for solution turbidity
[171. CD spectra were measured in a J41A JASCO instrument. Calibration of the instrument was done according to [ 181. The measurements were performed in cells 0.093,0.186,0.5 and 10.0 mm thick, in the far and near ultraviolet regions. For calculation of the ellipticity the mean molecular weight of an amino acid residue (MRW) was assumed to be 115.0 [2]. The estimation of the secondary structure content was done according to [ 191, using reported reference spectra [20].
September
1979
from the slope of the scattering curve in the Guinier coordinates (log I versus p2). The molecular weight was calculated from the curves as in [7].
3. Results 3 .l . Extinction coefficient and secondary structure Figure 1 presents the absorption spectrum of protein S15 in the ultraviolet region recorded in standard solvent. The extinction coefficient at 277 nm (A; “,p’“‘) is found to be 0.325 (*O.OlO). This extinction coefficient was used in all other experiments for determinations and checks of the protein concentration. It has been shown in special experiments that the absorption spectrum and the extinction coefficient do not change in solvents with pH values from 5.6 to 7 and salt concentration from 30 mM to 0.4 M. Figure 2 presents the CD spectrum of protein S15. It is seen that the spectrum corresponds to a highly a-helical protein. Estimation of the a-helical content in protein S15 from the CD spectrum [ 19,201 gives a value of 78%. The value of the helical content has been shown to remain unchanged, at least in the pH range 5.6 to 7 and salt concentration from 30 mM to 0.4 M. 3.2. Tertiary structure Figure 3 shows the PMR spectra of protein S15 in standard solvent (A) and in the presence of 5 M urea
2.3. PMR spectra Proton NMR spectra were recorded on a Bruker WH 360 spectrometer operating in the Fourier transform mode, using a pulse-length of 6 /LSwith 1.8 s intervals; the number of accumulations was 2000. Chemical shifts were measured relative to sodium 2,2-dimethyl-2-silapentane sulphonate as an internal standard. Spectra were obtained in 5 mm tubes with 0.8-I .O mg protein/ml in standard D20 solvent. 2.4. Neutron scattering Neutron scattering experiments were done in the Institut Laue-Langevin, Grenoble, on the high-flux reactor using the Dll camera [21] as in [7]. The cell was 2 mm thick with 0.75 mg protein/ml in standard DzO solvent. The radius of gyration was calculated 64
240
260
280
300
Wavelength,
320
340
360
nm
Fig.1. Absorption spectrum of protein S15 in the near ultraviolet region. Solvent: 0.1 M NaCl, 30 mM sodium phosphate (pH 5.6).
FEBSLETTERS
Volume 105, number 1
Wavelength,
September 1979
the spectrum of the protein under non-denaturing conditions (fig3A) contains wide overlapping resonance lines, mainly of apolar aliphatic residues and threonines. This indicates that the aliphatic side groups and the threonine residues are in different chemical surroundings within the protein, suggesting a specific folding of its chain. In the extreme high-field region, between 0.8 and 0.5 ppm, the PMR spectrum of protein S15 has a number of resonance lines with chemical shifts which are absent in individual amino acid residues [22]. These resonances can be attributed to aliphatic amino acid residues located near to aromatic amino acids in a globular structure [22,23]. In the low-field region, between 6.5 and 8.5 ppm, the resonance lines of 4 histidine, 2 tyrosine and 2 phenylalanine residues are revealed. Four separate signals, 2 protons each (at 6.79,6.85,7.09 and 7.14 ppm), have been attributed to the 2 tyrosine residues since they underwent a shift into the high field at pH >9.5 (data not shown). Inasmuch as the resonance lines at 6.79 and 7.09 ppm of one of the tyrosine residues are broader and their resolution poorer than the resonances at 6.85 and 7.14 ppm of the other, it can be suggested that one of the residues rotates around the Cfl-0 bond more freely than does the other [24].
nm
Wavelength, nm Fig.2. CD spectrum of protein S15 in the near (insertion) and the far ultraviolet regions. Solvent: 50 mM sodium phosphate (pH 5.6).
(B). Comparison of these spectra provides evidence that a well developed tertiary structure exists in protein S15 in standard solvent. In the high-field region, between 0.9 and 3.5 ppm,
His
C&i)
rn
7 mm
,
1
I
I
3
2
I
0
Fig.3. PMR spectra of protein S15. Solvent: 99.8% D,O with 0.1 M NaCl-30 mM sodium phosphate (pD 5.3) (A), and the same solvent in the presence of 5 M urea (B).
65
Volume 105, number 1
FEBS LETTERS
Protons at CZ of the imidazole rings of the 4 histidine residues of protein S15 under non-denaturing conditions exhibit separate signals in the region from 8.4 to 8.6 ppm indicating their different chemical surroundings, i.e., specific chain folding. Since their width is
